Mills will be able to access a more efficient and faster fermentation control system, avoiding production losses

Via a technique used in human laboratory tests, a group of researchers from the “Luiz de Queiroz” School of Agriculture at the University of São Paulo (ESALQ-USP) intends to improve the fermentation process of sugarcane biomass, which generates ethanol. “Bacteria and other microorganisms often interrupt the fermentation process. This implies huge losses of ethanol, which can come to five percent of the approximately 30 billion liters produced per year in Brazil,” says Engineer Agronomist Carlos Alberto Labate, Professor at ESALQ-USP and Coordinator of the Improving Ethanol Fermentation project.

The project was recently launched and will be developed over three years within the scope of the Research Centre for Innovation in Greenhouse Gases (RCGI), funded by the São Paulo Research Foundation (FAPESP) and by Shell of Brazil. The researchers will use Matrix-Assisted Laser Desorption ionization/Time-of-Light Mass Spectrometry (Maldi-TOF MS), which analyzes the structures of proteins found in cell membranes. “This technology was discovered in the 1990s, but about 15 years ago it began to be used in laboratory tests of patients hospitalized in Intensive Care Units (ICU) with serious and life-threatening infections. These people cannot wait for the result of the common bacterial culture test, which takes too long,” says Labate. “With the Maldi-TOF MS the result of the analysis of a small sample of the patient’s blood is processed in about 15 minutes.”

According to Labate, the technique consists of using a powerful laser beam to fragment the proteins present in the bacteria and fungi membranes into smaller pieces (peptides) and, thus, trace their amino acids. “In the medical field, the mass spectra of the peptides are immediately compared to more than 8,000 species of bacteria gathered in a database created by the manufacturer of the device,” he explains.

About five years ago, the researcher began using this equipment at the Plant Genetics Laboratory, which he heads at ESALQ-USP, for the purpose of analyzing the industrial fermentation of sugarcane. Since then, the laboratory team has been developing a database of samples collected at mills belonging to Raízen – a company located in outstate São Paulo and controlled by Shell of Brazil and Cosan S.A., which is a national leader in the manufacture of sugar, ethanol, and bioenergy. “We want to identify the microorganisms that are in the fermentation vats and take an X-ray of those contents.”

During the project, the researchers plan to expand the database (the collections will be conducted at two Raízen mills located in Piracicaba and Rafard in outstate São Paulo). “From the data collected in the field, we will train an artificial intelligence program to identify the best metabolic markers for fermentation. This information will become a part of the machine’s repertoire,” says Labate. “During the project, we will monitor three sugarcane crops. And that’s great, because the more information we get, the better it will be for the computer system.”

With this information in hand, the researchers will move on to another stage of the project. In a partnership with private enterprise, they will develop sensors which will be installed in the fermentation vats. “The sensors will work autonomously online, sending data in real time to the software especially tweaked by our team. Artificial intelligence will then record all the information, such as an abnormal increase in the number of bacteria in the fermentation process and, thus, generate a decision to help the mill’s technicians,” Labate states.

In his expert opinion, the research may bring greater security to the country’s ethanol fermentation process. “In Brazil, sugarcane mills started producing ethanol in the early 20th century, between wars, in order to take advantage of excess sucrose. That ethanol was generally sold for overall use in industry and homes. With the creation of the National Alcohol Program (Proálcool), in 1975, ethanol was converted into fuel and the mills turned their focus on this purpose, which is much more profitable. However, yet today, ethanol fermentation is conducted empirically, as it was in the beginning of the 20th century, where the vats are subject to contamination by bacteria and other microorganisms.”

Those invaders come from many sources, as is the case with wild yeasts. “The ethanol production process in Brazil uses commercial yeasts. However, it is common for wild yeasts, present in the biomass, to enter the system and dominate it, undermining the efficiency of the commercial yeast that is in the vat, thus disrupting the fermentation process,” says Labate. When such problems occur, mills often apply antibiotics to the liquid being fermented. “Sometimes this measure works, but, for the most part, it doesn’t, which makes the production of ethanol in that particular vat futile. So, the liquid is either discarded or used in the vinasse cycle to serve as a fertilizer,” he adds.

Labate warns that the process for identifying problems that affect the fermentation process is usually time consuming. “For example, the mills generally use the karyotype process to identify what type of yeast changed the balance of the microbiota (a set of bacteria, viruses, fungi, and other microorganisms) of the fermentation. The test takes about 15 days to complete. With the technology we are developing, the mills will be able to access a much more efficient and faster control system, allowing them to better control the fermentation process and avoid ethanol losses.”